PI3K/mTOR inhibition markedly potentiates HDAC inhibitor activity in NHL cells through BIM- and MCL-1-dependent mechanisms in vitro and in vivo

Abstract

Purpose: The aim of this study is to explore the efficacy and define mechanisms of action of coadministration of the PI3K/mTOR inhibitor BEZ235 and pan-HDAC inhibitor panobinostat in diffuse large B-cell lymphoma (DLBCL) cells.

Experimental design: Various DLBCL cells were exposed to panobinostat and BEZ235 alone or together after which apoptosis and signaling/survival pathway perturbations were monitored by flow cytometry and Western blot analysis. Genetic strategies defined the functional significance of such changes, and xenograft mouse models were used to assess tumor growth and animal survival.

Results: Panobinostat and BEZ235 interacted synergistically in ABC-, GC-, and double-hit DLBCL cells and MCL cells but not in normal CD34(+) cells. Synergism was associated with pronounced AKT dephosphorylation, GSK3 dephosphorylation/activation, Mcl-1 downregulation, Bim upregulation, increased Bcl-2/Bcl-xL binding, diminished Bax/Bak binding to Bcl-2/Bcl-xL/Mcl-1, increased γH2A.X phosphorylation and histone H3/H4 acetylation, and abrogation of p21(CIP1) induction. BEZ235/panobinostat lethality was not susceptible to stromal/microenvironmental forms of resistance. Genetic strategies confirmed significant functional roles for AKT inactivation, Mcl-1 downregulation, Bim upregulation, and Bax/Bak in synergism. Finally, coadministration of BEZ235 with panobinostat in immunocompromised mice bearing SU-DHL4-derived tumors significantly reduced tumor growth in association with similar signaling changes observed in vitro, and combined treatment increased animal survival compared with single agents.

Conclusions: BEZ235/panobinostat exhibits potent anti-DLBCL activity, including in poor-prognosis ABC- and double-hit subtypes, but not in normal CD34(+) cells. Synergism is most likely multifactorial, involving AKT inactivation/GSK3 activation, Bim upregulation, Mcl-1 downregulation, enhanced DNA damage, and is operative in vivo. Combined PI3K/mTOR and HDAC inhibition warrants further attention in DLBCL.

Fig. 1. Disruption of PI3K/AKT/mTOR pathway markedly potentiates panobinostat lethality in various NH lymphoma cell lines

(A-C) SU-DHL16 cells ectopically expressing a constitutively active AKT construct (A, inset) were exposed to increasing panobinostat concentrations for 24 hr, after which the extent of cell death was assessed using Annexin V/PI staining assay (A) and cell growth and viability were evaluated using a CellTiter-Glo Luminescent assay (B). Alternatively, Western blot analysis was performed following 4 hr treatment (C). (D-E) Cells were exposed to BEZ235 (25 nM for SU-DHL16 and 200 nM for the remaining cell lines) and panobinostat (7.5 nM for SU-DHL16; 10 nM for HBL-1, TMD8, OCI-LY18, CARNAVAL; and Jeko-1; and 15 nM for SU-DHL4, and OCI-LY7) alone or together for 24 hr for all cell lines with the exception of CARNAVAL cells (48 hr), after which cell death or cell growth and viability were assessed as in (A-B). (F-G) Western blot analysis on cytosolic fractions (F) or whole cell lysates (G) in various NHL cell lines following 24 hr treatment with BEZ235 and panobinostat as in D. For A, B, D, E, error bars = S.D for 3 independent experiments; * P < 0.02. C-PARP = cleaved PARP.

Fig. 2. Treatment with BEZ235/panobinostat is not toxic to normal human CD34⁺ cells, and is associated with a marked increase in histone H3 and H4 acetylation, induction of DNA damage, and abrogation of p21^CIP1 induction

Annexin V/PI analysis at 24 hr (A), or colony formation assay (B) on normal human CD34⁺ cells. C) Western blot analysis following 4-8 hr cell exposure to BEZ235 ± panobinostat.

Fig. 3. BEZ235/panobinostat lethality involves Mcl-1 down-regulation, and perturbations in Bim, Bax, and Bak

(A) Western blot analysis on cells treated for 8 hr with BEZ235 (200 nM) ± panobinostat (10 nM for HBL-1 and OCI-LY18; 15 nM for SU-DHL4). (B) Bax and Bak conformational changes in SU-DHL4 cells following 16 hr treatment as in A. C) cell growth and viability in SU-DHL4 and SU-DHL16 transiently transfected with siRNA against Mcl-1 (si-Mcl-1), or non-silencing siRNA (si-NS), and exposed to panobinostat for 16 hr. Error Bars: S.D of 3 independent experiments; * P < 0.05. D) SU-DHL4 and SU-DHL16 cells in which Bim was knocked down using lentiviral particles carrying shRNA constructs against Bim or non-silencing shRNA (sh-NS) were exposed to BEZ235 (200 nM for SUDHL4 and 25 nM for SU-DHL16) ± panobinostat (15 nM for SU-DHL4 and 7.5 nM for SU-DHL16) for 24 hr after which the extent of cell death was determined using Annexin V/PI assay. Error Bars: S.D of 3 separate experiments; * P < 0.01 for SU-DHL16 and P < 0.02 for SU-DHL4. E) Annexin V/PI assay in SU-DHL4 cells stably transfected with shRNA constructs against Bax (shBax), Bak (shBak), or non-silencing shRNA (sh-NS) following 24 hr treatment with 200 nM BEZ235 ± 15 nM panobinostat. Inset: Western blot on non-treated cells. Each sample was run twice. Error Bars: S.D of 3 separate experiments; * P < 0.05.

Fig. 4. Ectopic expression of constitutively active AKT or inhibition of GSK3 diminishes BEZ235/panobinostat lethality

A) Western blot on constitutively active Myc-tagged AKT-expressing SU-DHL16 cells following 24 hr exposure to 25 nM BEZ235 ± 7.5 nM panobinostat. Alternatively, the extent of cell death (B) or cell growth and viability (C) were assessed at 24 hr as in Fig. 1A-B. Error Bars: S.D of 3 independent experiments; * P < 0.02. D) SU-DHL4, SU-DHL16, HBL-1, and OCI-LY18 cells were treated with BEZ235 (200 nM for SU-DHL4, HBL-1, and OCI-LY18; 25 nM for SU-DHL16) ± panobinostat (7.5 nM for SU-DHL16; 10 nM for HBL-1, and OCI-LY18; 15 nM for SU-DHL4) for 8 hr, then cells were lysed and Western blot performed using the indicated antibodies. E) SU-DHL4 and HBL-1 cells were exposed to combined treatment with BEZ235 and panobinostat as in (D) in the presence or the absence of BIO, MeBIO, or CHIR-98014 (2 μM each). Twenty-four hr after treatment, cell growth and viability were assessed using CellTiter-Glo Luminescent assay. * = significantly greater than values obtained in the absence of BIO or CHIR-98014 (P < 0.05).

Fig. 5. BEZ235/panobinostat treatment is effective in the presence of a protective stromal microenvironment

A) SU-DHL4 cells were treated with 200 nM BEZ235 ± 15 nM panobinostat in the presence of HS-5-conditioned media (HS-5 CM) or regular media (RM) for 24 hr after which cells were lysed and subjected to Western blot analysis. B) Annexin V/PI assays in SU-DHL4, HBL-1, and OCI-LY18 cells following 24 hr treatment with BEZ235 (200 nM) ± panobinostat (10 nM for HBL-1 and OCI-LY18; 15 nM for SU-DHL4). C) SU-DHL4 and OCI-LY18 cells were co-cultured with HS-5 cells for 3 hr, and then exposed to BEZ235 ± panobinostat for 24 hr as in (B) after which cells were photographed using an Olympus microscope. D) Western blot analysis of SU-DHL4 cells treated as in (A) for 8 hr.

Fig. 6. BEZ235/panobinostat significantly inhibits tumor growth and prolongs survival in a xenograft mouse model

Tumor growth in beige nude mice (7 mice/condition) bearing luciferase-expressing SU-DHL4-derived tumors treated 5 days a week with BEZ235 (50 mg/kg) and panobinostat (15 mg/kg) alone or in combination. Mice were also imaged prior to and 3 weeks after treatment using an IVIS 200 imager (B). C) Xenograft-bearing mice were treated as in (A) twice over a 24 h interval after which tumors were excised, lysed, and subjected to Western blot analysis. D) Kaplan-Meyer plot involving 7 mice/condition treated as in (A). The median survival was prolonged from 34 to 51 days for mice exposed to the combined treatment. The survival curves differed significantly between combined treatment and other treatments (P = 0.001 for BEZ235/panobinostat vs panobinostat; P = 0.0386 for BEZ235/panobinostat vs BEZ235; logrank test).